Cumulative oxidative damage to tissues has been implicated in a number of disease states, e.g. the aging process, cancer, and ischemia reperfusion. The study of oxidative stress in the mitochondria has shown that hydrogen peroxide is produced via the incomplete reduction of oxygen during oxidative phosphorylation. Hydrogen peroxide levels are kept relatively low under normal physiological conditions. Under certain conditions, such as inflammation, excessive amounts of hydrogen peroxide is thought to precede several occurrences, such as lipid peroxidation, DNA and/or protein damage, and glutathione depletion, that are characteristic of oxidative stress. The role of free radicals in oxidative stress has led to an increased interest in the study of free radicals and their reactions. Reactive oxygen metabolites can interact with cellular constituents, including DNA/RNA, proteins, and unsaturated lipids. Previous studies have suggested that hemoproteins may be involved in redox reactions which contribute to tissue and/or organ damage via reaction with hydrogen peroxide. In addition, a recent study has reported evidence for the association between nitrotyrosine levels and coronary artery disease. Thus, the determination and characterization of protein radical intermediates is important in understanding the mechanisms of these reactions and their contributions to human diseases. The Mass Spectrometry Workgroup has had a long-term collaboration with Ronald Mason?s Free Radical Workgroup (LPC). The goal of this collaboration is to gain insight on the mechanisms of oxidative stress using a combination of spin trapping of oxidatively-induced free-radicals followed by structural characterization using mass spectrometry. The formation of radicals on horse myoglobin, sperm whale myoglobin, and hemoglobin was initiated by treating a solution of each protein with hydrogen peroxide in the presence of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). When either myoglobin or hemoglobin was reacted with hydrogen peroxide in the presence of DMPO, a DMPO nitrone adduct could be detected by immunostaining. To verify that DMPO adducts of the protein free-radicals had been formed, the reaction mixtures were analyzed by MS. These data showed an adduct of myoglobin which corresponds to the addition of one DMPO molecule. After digestion and MS/MS analysis, we determined that the DMPO was covalently bound to the Tyr-103 residue of the horse myoglobin. Concurrently, we have determined the site of DMPO adduct formation on sperm whale myoglobin to be Tyr-104. The nature of the radicals formed on hemoglobin has been explored using proteolysis techniques followed by LC/MS and MS/MS analyses. The following DMPO adducts have been confirmed on hemoglobin: Cys-93 of chain B, Tyr-42, Tyr-24 and His-20 of chain A. We have begun preliminary work to immobilize a monoclonal anti-DMPO antibody to sepharose beads to specifically pull out protein-DMPO and/or peptide-DMPO adducts on the proteins myeloperoxidase and lactoperoxidase. This technique should be useful in determining DMPO adducts on very large proteins and/or proteins from biological samples.